Abstract

It is unknown whether formoterol and salmeterol, two long-acting β2-adrenoreceptor agonists, have regulatory functions in the production of T-helper cell (Th) type 2- and Th1-related chemokines by monocytes and bronchial epithelial cells.

In the present study, the effects of formoterol and salmeterol on lipopolysaccharide (LPS)-induced expression of the Th2-related chemokine macrophage-derived chemokine (MDC; CCL22) and the Th1-related chemokine interferon-γ-inducible protein (IP)-10 (CXCL10) were investigated in a monocytic cell line, THP-1, and in human primary monocytes. In addition, their effects on the expression of the Th2-related chemokine thymus- and activation-regulated chemokine (TARC; CCL17) were evaluated in an epithelial cell line, BEAS-2B.

Formoterol enhanced MDC but suppressed IP-10 production in monocytes induced by LPS. Higher doses of salmeterol were required to enhance LPS-induced MDC expression in THP-1 cells. Formoterol and salmeterol could significantly suppress TARC expression in BEAS-2B cells. These effects could be reversed by a selective β2-adrenoreceptor antagonist, ICI-118551. Formoterol- and LPS-induced MDC expression was inhibited by budesonide.

Asthma is a chronic inflammatory disorder of the airway 1. Infiltration of the airways by T-helper cell (Th) type 2 lymphocytes, eosinophils and other inflammatory cells is a well-recognised feature of bronchial asthma 2, 3. The role of Th1-type responses in asthma has been of great interest, as there have been several proposed therapies and preventive measures attempting to reduce allergic airway inflammation by enhancing the Th1 inflammatory responses 2, 3. However, Th1 responses do not always downregulate allergic inflammation. Interferon (IFN)-γ-inducible protein (IP)-10 (CXCL10) is a chemokine that attracts Th1 lymphocytes through its receptor CXCR3 4. IP-10 is induced in a variety of cells in response to IFN-γ 4 and is also upregulated in allergic pulmonary inflammation 5. IP-10 is more highly expressed in airway smooth muscle of subjects with asthma when compared with healthy control subjects, suggesting that the CXCL10/CXCR3 axis may serve as a novel target for the treatment of asthma 6. Macrophage-derived chemokine (MDC; CCL22) and thymus- and activation-regulated chemokine (TARC; CCL17) are Th2 chemokines involved in the recruitment of CC chemokine receptor 4-bearing Th2 cells in allergen-induced inflammation 7. Increased levels of plasma MDC and TARC have been found in children with acute asthma, but their levels decrease after ketotifen treatment 8.

β2-Adrenoreceptor agonists are firstline drugs for the treatment of acute asthma due to their potent and rapid bronchodilatory effects. Inhaled long-acting β2-adrenoreceptor agonists (LABAs) are generally combined with corticosteroids to control asthma, and are considered to be smooth muscle relaxants. While their anti-inflammatory properties are still a matter of debate, their anti-inflammatory effects are suggested by inhibitory effects on granulocyte adhesion to epithelium 9 and on infiltration of inflammatory cells in the skin and lungs of guinea pigs 10. LABAs have also been shown to exert their inhibitory effects on the expression of the pro-inflammatory cytokines interleukin (IL)-6, IL-8 and tumour necrosis factor (TNF)-α in a variety of cell types. For example, formoterol has been shown to be able to suppress lipopolysaccharide (LPS)-induced IL-6 expression in a mouse model, but to enhance the expression of IL-8 in bronchial epithelial cells 11, 12. Also, salmeterol has been shown to inhibit TNF-α secretion in LPS-activated THP-1 cells 13 and to suppress the immunoglobulin E-dependent release of TNF-α from human skin mast cells 14. Wallin et al. 15 recently reported the inhibition of eosinophil infiltration by formoterol in subjects with asthma. Additionally, inhaled LABAs and corticosteroids give optimal control of asthma in most patients, and two fixed-combination inhalers (salmeterol/fluticasone and formoterol/budesonide) have increasingly been used as convenient controllers in patients with persistent asthma. Thus, there is a strong scientific rationale for the combination of these two drug classes 16.

In the present study, the authors investigated whether formoterol and salmeterol have regulatory effects on the expression of the Th2-related chemokines MDC and TARC and the Th1-related chemokine IP-10 in human monocytes (THP-1 cells and peripheral blood monocytes) and in bronchial epithelial cells (BEAS-2B).

Peripheral blood samples were obtained from three healthy individuals who had no personal or family history of allergic diseases. Cord blood was harvested from delivered placentas into a standard blood collection bag with citrate phosphate dextrose anticoagulant. The cord blood was collected from the delivered placenta to avoid any risk to the mother or infant from the collection process. The study protocol was approved by the Institutional Review Board of Kaohsiung Medical University Hospital (Kaohsiung, Taiwan, Republic of China). Informed consent was obtained from three healthy adult volunteers and each parent of the cord blood donors. Blood samples were diluted with an equal volume of PBS. Peripheral blood mononuclear cells were isolated by density gradient centrifugation (LymphoprepTM; Axis-Shield PoC, Oslo, Norway), and monocytes were isolated by magnetic bead sorting with the anti-CD14 monoclonal antibody (mAb) MACS® (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany).

Human mononuclear cells were isolated from the cord blood of healthy neonates using Ficoll-PaqueTM (GE Healthcare, Uppsala, Sweden). The CD4+ lymphocytes were further purified by magnetic bead sorting with an anti-CD4 mAb (MACS®; Miltenyi Biotec GmbH). The cells were polarised as previously described 18. Th2 cultures were supplemented with 10 μg·mL−1 anti-IL-12 (R&D Systems, Minneapolis, MN, USA) and 10 ng·mL−1 IL-4 (R&D Systems). T-cells were cultured with 5 ng·mL−1 IL-12 and 10 μg·mL−1 anti-IL-4 (R&D Systems) for Th1 polarisation. After 48 h of priming, 5 ng·mL−1 IL-2 (R&D Systems) was added to the cultures. The cells were cultured in the presence of IL-2 alone without the addition of any polarising cytokines. After 7 days of polarisation, IFN-γ and IL-4 cytokine production was determined by ELISA (R&D Systems; data not shown).

Chemotactic assay

Chemotaxis of Th1 and Th2 cells was measured using a 24-well Micro Chemotaxis Transwell (Corning Costar, Cambridge, MA, USA). Th1 and Th2 cells were resuspended at 3×105 cells·mL−1 and loaded onto the upper chamber of the Micro Chemotaxis chamber. The supernatants of LPS-treated THP-1 cells or IL-4-, TNF-α- and IFN-γ-treated BEAS-2B cells were added to the lower chamber. In some cases, both kinds of supernatant were mixed with a 1:1 ratio. The lower and upper chambers were separated by a polycarbonate membrane (5-μm pore size). Th1 and Th2 cells were left to transmigrate for 3 h at 37°C in a humidified atmosphere with 5% CO2. After incubation for 3 h, the number of migrated Th2 cells in the lower compartment was determined by counting the cells under a light microscope. The percentage of inhibition was calculated from three separate experiments.

Western blotting

After treatment for 2 h with or without salmeterol (10−8–10−6 M), the cells were stimulated with LPS (0.2 μg·mL−1) and lysed 1 h later with an equal volume (150 μL) of ice-cold lysis buffer. After centrifugation at 13,000×g for 15 min, equal amounts of cell lysates from each experimental condition were analysed by Western blot with anti-IκB-α and anti-β-actin antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). Immunoreactive bands were visualised using horseradish peroxidase-conjugated secondary antibodies and the enhanced chemiluminescence system (Amersham ECLTM; GE Healthcare).

RNA extraction and real-time PCR

Total RNA was extracted using RNeasy Mini Kits (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. From each sample, 3 μg RNA was reverse-transcribed to produce first-strand cDNA in a 20 μL reaction mixture using a SuperScriptTM First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA). Measurement was performed by an ABI PRISM® 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA) using a pre-designed TaqMan® probe/primer combination for TARC (Protech Technology, Taipei, Taiwan). TaqMan® PCR was performed in a 10 μL volume using AmpliTaq Gold® polymerase and universal master mix (Applied Biosystems). Threshold cycle numbers were transformed using the comparative threshold cycle and relative value method, as described by the manufacturer (Applied Biosystems), and were expressed relative to β-actin, which was used as a housekeeping gene by multiplexing single reactions.

ELISA assay

MDC, TARC and IP-10 concentrations of the cell supernatants were determined using commercially available ELISA-based assay systems (R&D Systems). Assays were performed using the protocols recommended by the manufacturer.

Statistical analysis

All data are presented as mean±sd. One-way ANOVA was used for all statistical comparisons, and the Student–Newman–Keuls test was conducted for multiple comparisons. A p-value <0.05 was considered statistically significant.

RESULTS

LPS-induced MDC production in THP-1 cells was significantly enhanced in the presence of formoterol (10−8–10−6 M) after 12 and 24 h of stimulation (fig. 1a⇓ and b). However, formoterol (10−8–10−6 M) alone could not induce MDC expression in THP-1 cells. Salmeterol also enhanced MDC production in THP-1 cells induced after 12 h of LPS stimulation, but only at the highest concentration (10−6 M; fig. 1a⇓). Similarly, formoterol and only a higher dose of salmeterol (10−6 M) were found to enhance TNF-α-induced MDC expression, and this enhanced expression was inhibited by budesonide (fig. 1c⇓). Moreover, the effect of formoterol was seen at the level of gene transcription, since formoterol and salmeterol (at 10−7 M) enhanced LPS-induced MDC mRNA expression in THP-1 cells (fig. 1d⇓). Interestingly, formoterol (10−9–10−7 M) but not salmeterol (10−9–10−7 M) enhanced the LPS-induced MDC production in human primary monocytes after 24 h of LPS stimulation (fig. 1e⇓).

Formoterol and salmeterol had no effect on LPS-induced MDC and IP-10 expression in THP-1-derived macrophages

Both LABAs and corticosteroids are usually administered locally in asthmatic patients, and macrophages are likely to be the targets for modulation. Using THP-1-derived macrophages, produced by in vitro culture of the cells with PMA, neither formoterol nor salmeterol had a significant effect on LPS-induced MDC, IP-10 or TNF-α expression (fig. 4⇓).

Formoterol and salmeterol suppressed TARC expression in BEAS-2B cells

When TARC expression in BEAS-2B cells was analysed, formoterol and salmeterol were shown to be able to suppress the TARC mRNA (fig. 5a⇓) and protein (fig. 5b⇓) expression in BEAS-2B cells stimulated with a combination of TNF-α (50 ng·mL−1), IL-4 (50 ng·mL−1) and IFN-γ (10 ng·mL−1). This suppressive effect was reversed by the addition of ICI-118551 (fig. 5c⇓). Both budesonide alone and the combination of budesonide and LABA significantly suppressed TARC expression in BEAS-2B cells (fig. 5d⇓).

Since LABAs have an opposite effect on the production of MDC by monocytes and of TARC by epithelial cells, analysis of the chemotactic activity of LABA-treated cells on Th2 and Th1 lymphocytes was performed. The results revealed that formoterol-treated THP-1 cells and BEAS-2B cells increased the chemotaxis of the Th2 cells but suppressed that of the Th1 lymphocytes (fig. 7⇓).

DISCUSSION

Chemokines are known to be important in the pathophysiology of asthma and allergies; in particular, increased expression of TARC and MDC has been found in the bronchial epithelium of asthmatic patients, and is associated with the recruitment of Th2 cells in the inflammatory airways 17, 19. Furthermore, LPS is known to be ubiquitously present in the environment and induces Th1- and Th2-related chemokine expression. Exposure to airborne endotoxin in infancy may protect against asthma by promoting the Th1 response and tolerance to allergens. However, it adversely affects asthma patients later in life 20. Salmeterol and formoterol have both been introduced as long-acting bronchodilators for the therapeutic management of asthma. In the present study, formoterol was shown to enhance the expression of a Th2-related chemokine, MDC, in LPS-treated monocytes, implying a possible occurrence of Th2-associated inflammation following formoterol monotherapy via systemic administration. Moreover, both LABAs were shown to be able to suppress the expression of TARC in bronchial epithelial cells at physiological concentrations. This may be the reason why formoterol and salmeterol should be administered in an inhalation form. Notably, LABAs showed opposite effects on the production of MDC in monocytes and of TARC in epithelial cells. Also, increased chemotactic activity of Th2 cells was found following treatment of the cells with LABA. It is possible, therefore, that the combined use of inhaled LABA and corticosteroid may provide an optimal control of asthma in most patients.

Additionally, chronic use of β2-adrenoreceptor agonists might increase the risk of bronchial hyperresponsiveness (BHR) and cause greater airway inflammation resulting in increased BHR and a greater decline in lung function 21. It has been suggested that β2-adrenoreceptor agonists may also mask the effects of increased airway inflammation 22. β2-Adrenoreceptors are present not only on smooth muscle cells but also on neutrophils, lymphocytes, monocytes and macrophages 23. In the present study, relevant drug concentrations mimicked the in vivo situation at the airway level (10−10–10−6 M formoterol and 10−8 M budesonide) 12. Formoterol at physiological concentrations enhanced LPS-induced MDC expression in human monocytes, which plays an important role in the pathogenesis of airway inflammation in asthma. Budesonide blocked formoterol and LPS-induced MDC expression. Therefore, the relevance of LABA monotherapy in asthma may be questioned, as it is advised that LABAs be used in combination with inhaled steroids, while short-acting β2-adrenoreceptor agonists should only be used without inhaled steroids in very mild intermittent asthma.

Formoterol is more potent and a full agonist, compared with salmeterol, which is a partial agonist 24. In addition, salmeterol is approximately two-thirds less efficacious than either formoterol or isoprenaline as an inhibitor of histamine release 24. In the present study, MDC production in monocytes induced by LPS was enhanced significantly by formoterol (10−9–10−7 M). Only higher doses of salmeterol (10−6 M), but not lower doses (10−9–10−7 M), could significantly enhance LPS-induced MDC expression in human monocytes. These results suggest, therefore, that the long-term use of a full LABA may have higher a risk of causing allergic inflammation in comparison with the use of a partial LABA. Formoterol is more potent in inducing the Th2-related chemokine MDC and suppressing the Th1-related chemokine IP-10, suggesting that the affinity for binding to the β2-adrenoreceptor may be the key point in inducing Th2- but suppressing Th1-related chemokines. β2-Adrenoreceptor agonists and budesonide could also suppress IL-1β release in blood monocytes; however, macrophages may exhibit more resistance to treatment with β2-adrenoreceptor agonists and budesonide 25. This may explain why monocyte-derived macrophages were not sensitive to the effect of LABAs on chemokine expression in the present study.

It is known that LABAs act primarily to relax airway smooth muscle via the activation of cAMP, and that additional effects of LABAs may include the stabilisation of inflammatory cell activity and inhibitory effects on the pulmonary mast cells, correlating with the increase in cAMP levels induced by these agonists 24, 26. To examine whether, indeed, increasing cAMP levels could enhance MDC expression in monocytes and suppress TARC expression in bronchial epithelial cells, THP-1 cells and BEAS-2B cells were pre-treated with the phosphodiesterase inhibitor etazolate for 1 h before stimulation. Etazolate enhanced LPS-induced MDC expression in THP-1 cells (fig. 6d⇑) but suppressed TARC expression in BEAS-2B cells (fig. 6e⇑). These results suggest that the expression of chemokines in monocytes and bronchial epithelial cells may be modulated, at least partially, via the β2-adrenoreceptor-cAMP pathway. Thus, in addition to their effects on relaxing airway smooth muscle and pulmonary mast cells, LABAs could also modulate Th1- and Th2-related chemokine production via the activation of cAMP.

NF-κB activation may be responsible, in part, for increased expression of many inflammatory genes in asthma 27. There has been conflicting evidence regarding the action of the cAMP/cAMP-dependent protein kinase (PKA) signalling pathway on NF-κB. It is known that PKA activating agents inhibit the NF-κB-dependent reporter gene expression induced by the activation of TNF-α 28. In macrophages, LPS-stimulated NF-κB has been shown to be cooperatively activated by cAMP-dependent and -independent PKA activation 29. In the present study, LABAs enhanced LPS-induced MDC expression in monocytes. ICI-118551, a selective β2-adrenoreceptor antagonist, reversed this effect. The finding that IκB-α expression was suppressed by salmeterol in the present study suggests that β2-adrenoreceptor-cAMP pathway activation may enhance LPS-induced NF-κB expression via suppression of IκB-α expression in monocytes. Within the proximal promoter region of MDC, potential binding sites for NF-κB subunits p50 and p65 have been identified 30. Thus, LABAs may suppress IκB-α expression and cause translocation of NF-κB into the nucleus by increasing cAMP and subsequently transactivating the promoter of the MDC gene.

In conclusion, through the nuclear factor-κB or the β2-adrenoreceptor-cyclic adenosine monophophate pathways, long-acting β2-adrenoreceptor agonists may increase T-helper cell type 2-related chemokine expression of monocytes and T-helper cell type 2 chemotaxis, but suppress T-helper cell type 2-related chemokine expression in bronchial epithelial cells. Therefore, long-acting β2-adrenoreceptor agonists may be better administered in an inhalation form and long-acting β2-adrenoreceptor agonist monotherapy may not be a good option for asthma.

Support statement

This study was supported by grant no. 95-2314-B-037-092 from the National Science Council, Republic of China.